Method for quickly homogenizing circular dna samples

ABSTRACT

The present application provides a method for quickly homogenizing circular DNA samples, comprising performing rolling circle amplification on circular DNAs in the samples, so that the concentration of the circular DNAs in the samples are homogenized.

TECHNICAL FIELD

The present invention relates to the field of biological engineering,and in particular, to a homogenization method suitable for samplescomprising circular DNAs. The method is specifically a method forquickly homogenizing circular DNA samples by a rolling circlereplication.

BACKGROUND ART

The experimental process of next-generation sequencing (NGS) mainlyinvolves three steps: sample template preparation, sequencing libraryconstruction, and on-line sequencing. Among these steps, the sequencinglibrary construction needs to control the initial sample amount ofdifferent detection samples, so that the concentrations of differentsamples are homogenized to facilitate downstream experimentaloperations. The conventional homogenization method (i.e.,quantification-calculation-pipetting) mainly estimates the amount ofDNAs contained through the absorbance value, so as to pipette the sameamount of samples. However, the method using the absorbance value orfluorescence quantification will be affected by other proteins, othertypes of nucleic acids or impurities with similar absorption of specificspectra, and the fluorescence quantification has the disadvantages ofhigh cost, tedious and time-consuming operation, etc. Specifically, theoperation time of homogenizing 10,000 samples is as follows: (1) for thecalculation step, it takes about 100 hours to input the concentration ofeach sample and calculate the specific pipetted sample amount; and (2)for the process of adjusting a pipette and pipetting the correspondingcalculated amount of samples separately from each sample to achievehomogenization between samples, it takes 100 hours. Therefore, accordingto the existing technical process, the entire homogenization processrequires a manual operation time of 200 hours.

The rolling circle amplification (RCA) technology can be used to amplifycircular DNA templates, such as plasmids and phage DNAs. This technologyis a nucleic acid amplification technology established on the basis ofthe method of rolling circle replication of circular DNA molecules ofpathogenic microorganisms in nature, and it is a DNA amplificationtechnology that occurs at a constant temperature (Lizardi et al., 1998).

An RCA system comprises: (1) phage phi29 DNA polymerase; (2) a circulartemplate (currently the RCA amplification template may also be linear);and (3) primers, which generally need to be resistant to endonuclease.Among these, the phage phi29 DNA polymerase is very important. It hashigh strand displacement activity, and can perform 70 kb stranddisplacement DNA synthesis without template separation. Moreover, it hasa stable performance and a high continuous synthesis capacity, and canefficiently catalyze DNA synthesis for several hours at a synthesisspeed of about 50 bp/s. In addition, the phi29 DNA polymerase has strongerror correction ability, and the error rate is comparable to that ofpfu enzyme, and significantly lower than that of Taq DNA polymerase. RCAis an isothermal signal amplification method with a linear amplificationfactor of 10⁵ and an exponential amplification capacity of greater than10⁹. The RCA technology is a trace molecular detection method that canbe used for the detection and research of extremely trace biologicalmacromolecules and biomarkers. At present, this method has beensuccessfully used for the amplification of linear double-stranded DNAsand even whole genomic DNAs (Esteban et al., 1993; Qian et al., 2003;Simison et al., 2006). Therefore, the RCA technology, with its uniqueadvantages, provides the possibility of sample homogenization before theNGS library construction.

The present invention optimizes the rolling circle amplification (RCA)technology, and uses it to amplify circular DNA templates, such asplasmids and phage DNAs, to homogenize different concentrations ofcircular DNA samples, so that the coefficient of variation of theconcentrations of DNA templates of different samples is lower than 0.1,and the manual operation time is less than 5 hours.

SUMMARY OF THE INVENTION

An objective of the present invention is to solve the problems of highcost and a long consuming time for homogenizing large quantities ofsamples in the preparation of DNA libraries, especially in thepreparation of sequencing libraries for NGS.

In order to solve this problem, in one aspect, the present applicationprovides a method for homogenizing circular DNA samples based on a rapidrolling circle amplification, comprising: performing rolling circleamplification on each sample, so that the concentrations of the circularDNAs in the samples are homogenized.

According to some embodiments, the homogenization method comprises astep of performing denaturing treatment on each sample before performingthe rolling circle amplification. In the context of the presentapplication, “denaturing” is understood in accordance with the ordinarymeaning of nucleic acid denaturation in the art. According to somefurther embodiments, the denaturing treatment comprises treating thesamples at an elevated temperature until the denaturation is completed,and then lowering the temperature of the samples. According to some morespecific embodiments, the denaturing treatment is performed by heatingthe samples to a temperature of about 95° C. for 3 minutes. According tosome more specific embodiments, the samples are cooled to 4° C. afterheating, and kept. According to some further embodiments, random primersfor the rolling circle amplification are added to a reaction system ofthe denaturing treatment. In some specific embodiments, the randomprimers are 7 bp in length. Without wishing to be bound by any theory,the high temperature can ensure the sufficient release of the initialtemplate DNAs, and the cooling process can allow the random primer(s)and the circular DNA template to be annealed. By adding random primersbefore the denaturation process, compared with addition of randomprimers after the denaturation process, the effect of homogenizingcircular DNAs by rolling circle amplification can be further improved.

In some embodiments, the reaction system of the denaturing treatmentcomprises EDTA. In some preferred embodiments, the concentration of EDTAin the reaction system of the denaturing treatment is about 0.05 mM. Insome more specific embodiments, the denaturing treatment preferablycomprises the steps of: to 2 μl of an amplification template sample,adding 5 μl of a denaturing buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0,25° C.), and adding 3 μl of random primers (100-200 uM), mixing well,treating at 95° C. for 3 minutes to complete the denaturation, andcooling to 4° C.

In some embodiments, the reaction system of the rolling circleamplification comprises phi29 DNA polymerase. In some furtherembodiments, the rolling circle amplification comprises an isothermalamplification at a temperature of 30° C. for 3 hours. In some furtherembodiments, the reaction system of the rolling circle amplificationcomprises bovine serum albumin (BSA). In some further embodiments, theconcentration of bovine serum albumin (BSA) is about 0.1 mg/mL. In someembodiments, the reaction system of the rolling circle amplificationcomprises KCl. In some further embodiments, the concentration of KCl inthe reaction system of the rolling circle amplification is about 75 mM.

According to a specific embodiment of the method of the presentinvention, the rolling circle amplification preferably comprises thesteps of: to the product which has been subjected to the step ofdenaturing treatment, adding 2 μl of 10×amplification buffer (500 mMTris-HCl, 50 mM MgCl₂, 750 mM KCl, 50 mM DTT, pH 8.2, 25° C.), 0.2 μl ofBSA (10-50 mg/ml), 2.4 μl of dNTPs (2.5-10 mM), 3 μl of phi29 DNApolymerase (5-10 U/μl), and 2.4 μl of ddH₂O, made up to 20 μl of thefinal total reaction system, mixing well, amplifying at 30° C. for 4hours, treating at 65° C. for 10 minutes, and cooling to 4° C.

In a preferred embodiment, the homogenization method of the presentinvention further comprises treating the amplification product with anendonuclease after the completion of the rolling circle replication andamplification. In a further embodiment, the endonuclease is a BamH1-HFendonuclease.

In a preferred embodiment, the homogenization method of the presentinvention comprises neither a step of quantifying an initial amount ofthe circular DNAs in the samples, nor a step of calculating a sampleamount to be pipetted based on the measured amount of the circular DNAsin the samples.

The sample applicable to the present invention may be any biologicalsample comprising or consisting substantially of circular DNAs, forexample, without limitation, bacterial solutions, colonies, cell debris,plasmids, M13 phage, and any other circular DNA samples. The sample mayor may not be purified before being applied to the method of the presentinvention.

The method of the present invention can be used in any application whereit is desired to homogenize a plurality of samples containing circularDNAs, including, but not limited to, the preparation of DNA libraries,especially DNA libraries for next-generation sequencing (NGS).

Therefore, in one aspect, the present application provides a method forpreparing a DNA library from a plurality of samples containing circularDNAs, comprising treating each sample using the homogenization method ofthe present invention. In a preferred embodiment, the method forpreparing a library comprises pipetting the same amount of samples toprepare a library after treating a plurality of samples using thehomogenization method of the present invention.

In another aspect, the present application provides a next-generationsequencing method, comprising treating a plurality of samples to besequenced using the homogenization method of the present invention toprepare a sequencing library, and performing a next-generationsequencing on the sequencing library. The sequencing method can be usedon any next-generation sequencing platform, including, but not limitedto, Roche 454 platform, Illumina Solexa platform, ABI SOliD platform,etc.

Beneficial Effects

The rolling circle amplification method used in the present invention isgenerated by optimizing based on the commercial reagents and the rollingcircle amplification method reported in reference documents. Thisrolling circle amplification method optimizes the existing rolling ringamplification system, reagents and method flow as a whole; andeffectively solves the problem of circular DNA homogenization,especially the circular DNA homogenization before the large-scale NGSlibrary construction, on the premise of ensuring that the libraryconstruction effect and sequencing effect are comparable to the effectsin the case of a traditional DNA homogenization. Conventional DNAhomogenization of 10,000 samples requires a process ofquantification-calculation-pipetting, and it will take 200 hours toperform the above operation on a large-scale sample. Since the method ofthe present invention does not require the operation ofquantification-calculation-pipetting, an automatic pipetting platformcan be used to complete the pretreatment-homogenization process of10,000 NGS library samples within 4.5 hours, which greatly shortens thetime of sample pretreatment. In general, the method for homogenizing theNGS library samples based on rolling circle amplification provided bythe present invention solves the shortcoming of time consuming andtedious operation in the sample homogenization at an early stage of thelarge-scale NGS library construction, and its unique design can realizethe early-stage homogenization of NGS library samples quickly andeasily.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flow chart of homogenization of circular DNAs of thepresent invention.

DETAILED DESCRIPTION OF EMBODIMENTS

In order to further understand the method described in the presentinvention, the present invention will be further described below withreference to the accompanying drawings and examples.

Example 1

This example compares the homogenization effect (i.e. the coefficient ofvariation quantified by Qubit) of the concentrations of DNA templatesprepared by three different rolling circle amplification methods withinthe same time:

A. Related procedures for NEB phi29 DNA polymerase reagent (Cat #M0269S,this reagent provides some supporting reagents and experimentalprotocols for RCA reactions) (hereinafter referred to as “NEB reagent(phi29 DNA polymerase)”)

B. RCA experimental protocol cited from literature (Frank B. Dean., JohnR. Nelson., Theresa L. Giesler., and Roger S. Lasken. 2001. RapidAmplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase andMultiply-Primed Rolling Circle Amplification. Genome Research.1095-1099) (hereinafter referred to as the “methods in the literature”)

C. The homogenization method provided by the present application(hereinafter referred to as “the present application”)

The test samples were 16 bacterial solutions used in actual productionwhich were transformed with plasmids. The OD600 value of each bacterialsolution was measured with a spectrophotometer, and the results areshown in Table 1.

Three different sequencing template preparation processes were used toprepare sequencing templates, as described below:

1) Step 1 (Denaturation Process): Performing Denaturation and RandomPrimer Annealing on a Bacterial Solution Template

The experimental samples were bacterial solutions transformed withplasmids as described above. The system configurations of denaturationreactions for three different sequencing template preparation methodsare listed in the following table respectively:

Component Volume (μl) NEB reagent (phi29 DNA polymerase) Bacterialsolution 1 10x reaction buffer 1 (500 mM Tris-HCl, 100 mM MgCl₂, 100 mM(NH₄)₂SO₄, 40 mMDTT, pH 7.5, 25° C. □) 7 bp random primer (100 uM) 2.5ddH₂O 4.3 Total volume 8.8 Methods in the literature Bacterial solution2 TE buffer (10 mM Tris-HCl, 8 1 mM EDTA, pH 8.0, 25° C.) Total volume10 The present application Bacterial solution 2 Denaturing buffer 5 (10mM Tris-HCl, 0.1 mM EDTA, pH 8.0, 25° C.) 7 bp random primer (175 uM) 3Total volume 10

Three different sequencing template preparation methods used the sameexperimental operation flow of denaturation reaction, as shown in thefollowing table:

Temperature Time (min) 95° C. 3 4° C. Keeping

2) Step 2 (Amplification Process): Performing Isothermal Rolling CircleAmplification on Target Plasmid after Denaturation and Annealing

Using the reaction product from Step 1 as an experimental sample, theisothermal rolling circle amplification was performed according to threedifferent sequencing template preparation methods. The systemconfiguration of the amplification reaction in each method is shown inthe following table:

Component Volume (μl) NEB reagent (phi29 DNA polymerase) Reactionproduct from Step 1 8.8 dNTP (10 mM) 0.5 BSA (10 mg/mL) 0.2 Phi29 DNApolymerase (10 U/μl) 0.5 Total volume 10 Methods in the literatureReaction product from Step 1 10 10x amplification buffer 2 (500 mMTris-HCl, 50 mM MgCl₂, 750 mM KCl, 1 mM DTT, pH 8.2, 25° C.) 7 bp randomprimer (100 uM) 1 dNTP (2.5 mM) 0.8 Phi29 DNA polymerase (10 U/μl) 0.5Yeast pyrophosphatase (0.1 U/μl) 0.3 ddH₂O 5.4 Total volume 20 Thepresent application Reaction product from Step 1 10 10x reaction buffer(500 mM 2 Tris-HCl, 50 mM MgCl₂, 750 mM KCl, 50 mM DTT, pH 8.2, 25° C.)BSA (10 mg/mL) 0.2 dNTP (3 mM) 2.4 Phi29 DNA polymerase (5 U/μl) 3 ddH₂O2.4 Total volume 20

Three different sequencing template preparation methods used the sameexperimental operation flow of isothermal amplification, as shown in thefollowing table:

Temperature Time 30° C. 4 hours 65° C. 10 minutes 4° C. Keeping

3) RCA Product Treatment and Quantification

The RCA product has a high-order structure; therefore, in this example,all the RCA products were enzymatically digested with BamH1-HF to makethe quantification more accurate. The NEB reagent (phi29 DNA polymerase)was configured by a digestion reaction system, and the methods in theliterature and the method provided in the present application wereconfigured by another enzymatic digestion reaction system, as describedin the following table:

Component Volume (μl) NEB reagent (phi29 DNA polymerase) 10x Cut SmartBuffer (Cat# B7203S, 3 500 mM potassium acetate 200 mM Tris-acetic acid100 mM magnesium acetate 1,000 ug/ml BSA pH 7.9, 25° C.) RCA product 10BamH1-HF (2 U/μl, Cat# R3136S) 1 ddH₂O 16 Total volume 30 Methods in theliterature/The present application 10x Cut Smart Buffer (Cat# B7203S, 3500 mM potassium acetate 200 mM Tris-acetic acid 100 mM magnesiumacetate 1,000 ug/ml BSA pH 7.9, 25° C.) RCA product 20 BamH1-HF (2 U/μl,Cat# R3136S) 1 ddH₂O 6 Total volume 30

The experimental operation flow of digestion reaction in three differentsequencing template preparation methods is consistent:

Temperature Time 37° C. 1 hour 65° C. 20 minutes 4° C. Keeping

4 μl of each sample after the above reaction was quantified using aQubit™ fluorescence quantifier (Cat #Q33216, Thermo Fisher). The resultsare shown in Table 2. Based on the raw data in Tables 1 and 2, thecoefficients of variation (ratio of standard deviation to mean) of theOD600 measurement of each initial bacterial solution, and of the Qubitvalues of the DNA templates respectively prepared by using the NEB phi29DNA polymerase kit, the method in the literature, and the method of thepresent application in Table 2 were calculated, respectively. Theresults are shown in Table 3.

Results

In this example, the use of the method provided in the presentapplication successfully achieves the homogenization of circular DNAsamples. Meanwhile, we compared the differences among the method of NEBreagent product and the method reported in the literature and the methodof the present application. The coefficient of variation of the OD600measurement (see Table 1) before treatment on an initial bacterialsolution was 0.408 (see Table 3); the coefficient of variation of theQubit value (see Table 2 for details) of the DNA template obtained bytreating with NEB reagent was 0.174 (see Table 3); and the coefficientof variation of the Qubit value (see Table 2) of the DNA templateobtained by treating with the RCA method reported in the literature was0.12 (see Table 3). The coefficient of variation of the Qubit value (seeTable 2) of the DNA template obtained by the method of the presentapplication was 0.086 (see Table 3). It can be seen that thecoefficients of variation of the concentrations (see Table 2) of the DNAtemplates obtained after three treatments are all lower than thecoefficient of variation of the OD value of the initial bacterialsolution, and the effect of homogenizing the content of circular DNAs inthe samples is achieved. In particular, the coefficient of variation ofthe sample obtained by the method of the present application issignificantly lower than that of the sample obtained by the NEB reagenttreatment and the method in the literature, indicating that the methodprovided by the present application further improves the homogenizationeffect of DNA samples.

TABLE 1 OD600 measurements of the bacterial solution samples of Example1 Bacterial Solution OD600 Bacterial solution 1 0.269 Bacterial solution2 0.38 Bacterial solution 3 0.264 Bacterial solution 4 0.331 Bacterialsolution 5 0.308 Bacterial solution 6 0.277 Bacterial solution 7 0.772Bacterial solution 8 0.305 Bacterial solution 9 0.308 Bacterial solution10 0.286 Bacterial solution 11 0.291 Bacterial solution 12 0.305Bacterial solution 13 0.658 Bacterial solution 14 0.255 Bacterialsolution 15 0.354 Bacterial solution 16 0.341

TABLE 2 Qubit measurements of the amplified products obtained in eachexperimental method of Example 1 RCA product concentration (ng/μL, afterenzymatic digestion) NEB phi29 DNA polymerase Methods The reagent in thepresent Bacterial solution (Cat# M0269S) literature invention Bacterialsolution 1 39.4 6.1 29 Bacterial solution 2 39 7 35.5 Bacterial solution3 44.2 5.7 29.7 Bacterial solution 4 42.4 6.75 32.7 Bacterial solution 542.8 6.75 33.3 Bacterial solution 6 43 6.6 31.2 Bacterial solution 739.4 6.2 30.8 Bacterial solution 8 38.8 7.3 34.4 Bacterial solution 918.6 7.55 31.3 Bacterial solution 10 33.6 6.6 33 Bacterial solution 1147.4 5.7 31.8 Bacterial solution 12 39.4 6.35 32.9 Bacterial solution 1347 5.4 26.9 Bacterial solution 14 48.1 5.15 26.6 Bacterial solution 1545 7.5 35.8 Bacterial solution 16 38.2 7.7 33.8

TABLE 3 Comparison table for coefficients of variation (CV) of thesample concentrations of Example 1. Coefficient of Type of measurementdata variation (CV) OD600 value of the initial bacterial solution 0.408Qubit value of the DNA template prepared by 0.174 homogenization usingthe NEB phi29 DNA polymerase kit Qubit value of the DNA templateprepared by 0.12 homogenization using the method in the literature Qubitvalue of the DNA template prepared by 0.086 homogenization using themethod of the present invention

In addition, the present invention is easy to use in connection with anautomatic pipetting platform. Taking Example 1 as an example, aftercombining the present invention with an automatic pipetting platform,the time taken to homogenize 10,000 samples using the method of thepresent invention can be controlled within 5 hours; while the time takenby the conventional homogenization method (i.e.quantification-calculation-pipetting) increases linearly with theincrease of the sample amount, and for the homogenization of 10,000samples to be treated, it takes up to about 200 hours.

1. A method for homogenizing a plurality of samples comprising circularDNAs, comprising: performing rolling circle amplification on circularDNAs in the samples, so that the concentrations of the circular DNAs inthe samples are homogenized.
 2. The method of claim 1, wherein themethod further comprises performing denaturing treatment on each samplebefore performing the rolling circle amplification.
 3. The method ofclaim 2, wherein the samples are mixed with random primers for therolling circle replication before starting the denaturing treatment. 4.The method of claim 2, wherein the denaturing treatment comprisesheating the samples to 95° C. and keeping for 3 minutes, and thencooling to 4° C. and keeping.
 5. The method of claim 2, wherein a systemof the denaturing treatment comprises EDTA.
 6. The method of claim 1,wherein a reaction system of the rolling circle amplification comprisesbovine serum albumin.
 7. The method of claim 1, wherein the reactionsystem of the rolling circle amplification comprises KCl.
 8. The methodof claim 1, wherein the method comprises neither a step of quantifyingan initial amount of the circular DNAs in the samples, nor a step ofcalculating a sample amount to be pipetted based on the measured amountof the circular DNAs in the samples.
 9. A method for preparing a DNAlibrary from a plurality of samples comprising circular DNAs, comprisingtreating the samples using the method of claim
 1. 10. A next-generationsequencing method, comprising: treating a plurality of samples to besequenced using the method of claim 1 to prepare a sequencing library,and performing a next-generation sequencing on the sequencing library.11. The method of claim 5, wherein the system of the denaturingtreatment comprises about 0.05 mM EDTA.
 12. The method of 6, wherein thereaction system of the rolling circle amplification comprises about 0.1mg/mL bovine serum albumin.
 13. The method of claim 7, wherein thereaction system of the rolling circle amplification comprises about 75mM KCl.
 14. The method of claim 6, wherein the reaction system of therolling circle amplification further comprises KCl.
 15. The method ofclaim 14, wherein the reaction system of the rolling circleamplification further comprises about 75 mM KCl.